Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization wit. h 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase 11 or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-p-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6 beta 3(1 -> 3)gal, accounting for approximate to 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase 11) from Bacillus sp. generates two major products, the monosulfated disaccharide gal beta(1 -> 4)glcNAc6s (approximate to 50% nmol product) and the disulfated disaccharide gal6s beta(I -> 4)glcNAc6s (approximate to 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximate to 108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide gal beta(I -> 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6s beta(I -> 4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80 +/- 0.34 mu g/ml/ 106 cells and composed of approximate to 71%nmol of disaccharide gal beta(1 -> 4)glcNAc6s and 18%nmol of the disulfated disaccharide gal6s beta(1 -> 4)glcNAc6s having approximate to 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.